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Abstract:

Compositions for the treatment of renal diseases and disorders utilize
agents which inhibit alphaV integrin molecules in vivo. Methods of
treatment include use of these agents in the prevention and treatment of
proteinuria, progressive glomerular disease and glomerular disease
amongst others.

Claims:

1. A method of reducing proteinuria in a subject, the method comprising
the step of administering to the subject an amount of a pharmaceutical
composition comprising an antibody that specifically binds
αVβ3 integrin and/or αVβ5 integrin, in
therapeutically effective amounts to reduce proteinuria in a subject.

2. The method of claim 1, wherein the antibody is a monoclonal or
polyclonal antibody.

3. The method of claim 1, wherein the antibody is a human antibody, a
humanized antibody, or fragments thereof.

4. The method of claim 1, wherein the antibody is CNTO 95.

5. The method of claim 2, wherein the proteinuria in a patient is reduced
by at least about 20% as measured by the patient's urinary protein
concentrations.

7. A method comprising the steps of (a) identifying a subject with
proteinuria; (b) administering to the subject a pharmaceutical
composition comprising a monoclonal antibody that specifically binds
αVβ3 integrin; and then (c) analyzing urinary protein
concentration in the subject.

8. The method of claim 7, further comprising the step of:
re-administering to the subject the pharmaceutical composition.

9. The method of claim 7, wherein the antibody is a human or humanized
antibody.

10. The method of claim 7, wherein the antibody is CNTO 95.

11. The method of claim 7, wherein the proteinuria is reduced by at least
20% after the step of administering the pharmaceutical composition.

23. The pharmaceutical composition of claim 20, wherein the agent is an
antibody.

24. A method of preventing or treating kidney disease or disorders in
vivo, comprising administering to a patient an agent in a therapeutically
effective amount, whereby the agent modulates the expression, function or
signaling of alphaV integrin molecules in vivo as measured by a decrease
in the patient's urinary protein concentration; and, preventing or
treating kidney disease in vivo.

32. The method of claim 31, further comprising the step of:
re-administering to the subject the pharmaceutical composition.

33. The method of claim 31, wherein the agent is an antibody, or a
fragments thereof.

34. The method of claim 31, wherein the urinary protein concentration in
the subject is reduced by at least 20% as compared to a normal control
after the step of administering the pharmaceutical composition.

[0002] Embodiments of the invention comprise compositions which prevent or
treat renal diseases and methods of use.

BACKGROUND

[0003] Integrins are a superfamily of cell adhesion receptors, which exist
as heterodimeric transmembrane glycoproteins. They are part of a large
family of cell adhesion receptors which are involved in
cell-extracellular matrix and cell-cell interactions. Integrins play
critical roles in cell adhesion to the extracellular matrix (ECM) which,
in turn, mediates cell survival, proliferation and migration through
intracellular signaling. The receptors consist of two subunits that are
non-covalently bound. Those subunits are called alpha and beta. The alpha
subunits all have some homology to each other, as do the beta subunits.
The receptors always contain one alpha chain and one beta chain and are
thus called heterodimeric. Both of the subunits contribute to the binding
of ligand. Eighteen alpha subunits and eight beta subunits have been
identified, which heterodimerize to form at least 24 distinct integrin
receptors.

[0004] Among the variety of alpha chain subunits is a protein chain
referred to as alpha V. The ITAGV gene encodes integrin alpha chain V
(alphaV). The I-domain containing integrin alpha V undergoes
post-translational cleavage to yield disulfide-linked heavy and light
chains, that combine with multiple integrin beta chains to four different
integrins. Alternative splicing of the gene yields 7 different
transcripts; a, b, c, e, f, h, j altogether encoding 6 different protein
isoforms of alphaV. Among the known associating beta chains (beta chains
1, 3, 5, 6, and 8; `ITGB1`, `ITGB3`, `ITGB5`, `ITGB6`, and `ITGB8`), each
can interact with extracellular matrix ligands. The alpha V beta 3
integrin, perhaps the most studied of these, is referred to as the
vitronectin receptor (VNR). In addition to providing for cell attachment
to other cells or to extracellular proteins such as vitronectin
(alphaVbeta3) and fibronectin (alphaVbeta6), the integrins are capable of
intracellular signaling which provides clues for cell migration and
secretion of or elaboration of other proteins involved in cell motility
and invasion and angiogenesis. The alpha V integrin subfamily of
integrins recognize the ligand motif Arg-GlyAsp (RGD) present in
fibronectin, vitronectin, Von Willebrand factor, and fibrinogen.

SUMMARY

[0005] This Summary is provided to present a summary of the invention to
briefly indicate the nature and substance of the invention. It is
submitted with the understanding that it will not be used to interpret or
limit the scope or meaning of the claims.

[0006] Embodiments of the invention are directed to compositions for the
treatment renal diseases or disorders, such as for example, proteinuria.

[0007] Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 is a schematic outlining a normal filtration barrier (a) and
an impaired barrier (b) in glomerular disease that is characterized by
foot process effacement. On a mechanistic level, the urokinase receptor
can associate with podocyte integrins (in particular beta3 integrins) and
increase integrin activity. This step is critical for foot process
effacement and the development of proteinuric kidney disease.

[0009] FIG. 2 is a graph showing the treatment of kidney disease. CNTO95
was administered in escalating dosages (ranging from 10 mg/kg to 100
mg/kg) intravenously (for small dosages, the injection volume was topped
up to 500 μl with PBS; for large dosages when the CNTO95 volume is
more than 500 μl, the actual required volume was injected). The first
injection of CNTO95 was given 1 hour prior to the injection of Puromycin
aminonucleoside (PAN). Additional injections were given on day 2, 4, 6,
8, 12, and 21. 8 days after induction of proteinuric kidney disease by
PAN, there was a significant reduction of proteinuria by up to 27%
(p<0.05). The day 8 timepoint is considered the peak phase of
proteinuria. CNTO95 is only poorly reactive in rats (please see Kd
values) yet still is associated with a significant reduction in
proteinuria.

[0010] FIG. 3 is a graph showing CNTO95 specific for the alphaVbeta3
integrin reduces proteinuria in PAN rats. CNTO95 was administered
intravenously at a volume of 500 μl 1 hour prior to the injection of
Puromycin aminonucleoside. This set up is a preventive set-up. 8 days
after induction of proteinuric kidney disease, there was a significant
reduction of proteinuria by up to 27% (p<0.05). The 7-8 day timepoint
is considered the peak phase of proteinuria. CNTO95 is only poorly
reactive in rats (please see Kd values) yet still is associated with
a significant reduction in proteinuria.

[0011] FIG. 4 is a graph showing baseline proteinuria of rats before
receiving CNTO95 and/or PAN. Rats show comparable levels of minimal
baseline proteinuria (left panel). CNTO95 was administered intravenously
at a volume of 500 μl 1 hour prior to the injection of Puromycin
aminonucleoside. This set up is a preventive set-up. 8 days after
induction of proteinuric kidney disease, there was a significant
reduction of proteinuria by up to 27%. The day 8 timepoint is considered
the peak phase of proteinuria. CNTO95 is only poorly reactive in rats
(please see Kd values) yet still is associated with a significant
reduction in proteinuria.

[0012] FIG. 5 is a graph showing the effects of CNTO95 administration 6
weeks after PAN induced glomerular proteinuria. A 38% reduction of
proteinuria (p<0.05) was noted. Furthermore, one rat died in the
non-CNTO95 group.

[0013] FIG. 6 shows the results of immunofluorescence after incubation of
CNTO95 with differentiated human podocytes in cell culture model. The
staining in green comes from immunofluorescent labeling of active beta3
integrins using AP5 antibody. Under control conditions, there is a low
baseline AP5 labeling. It is however much increased after 24 hours of PAN
treatment (see also Wei et al. Nat. Med. 2008). PAN was given 4 hours
prior to CNTO95 (treatment approach) and then left active for another 20
hours (together with CNTO95 at 1 microgram/ml). A reduction of AP5 signal
was noted indicating reduction in beta3 integrins (such as alphavbeta3 or
alphavbeta5). The middle panel shows human podocytes treated with
different dosages of CNTO95 from 1 μg/ml to 10 μg/ml. AP5 labeling
starts to increase at high dosages of CNTO95 which is most likely due to
clustering of beta3 integrins induced by CNTO95 in high concentration.
Lower panel: Podocytes under normal conditions, and after stimulation
with soluble uPAR as well as treated with soluble uPAR plus CNTO95 (1
microgram/ml) were compared for AP5 labeling. suPAR induced the AP5 label
but not in the presence of CNTO95 (1 microgram/ml).

[0014] FIG. 7 is a scan of a photograph of an immunostain showing the
activity of beta3 integrin in podocytes (using AP5 antibody) in human
Diabetic Nephropathy stages CKD 2-4.

DETAILED DESCRIPTION

[0015] Embodiments of the invention relate to discoveries involving agents
which modulate and/or inhibit the function, expression, activity or
combinations thereof, of alphaV (aV) integrins. Modulation of the alpha V
integrins are directed to treatment of kidney diseases or disorders such
as, for example, proteinuria.

[0016] Several aspects of the invention are described below with reference
to example applications for illustration. It should be understood that
numerous specific details, relationships, and methods are set forth to
provide a full understanding of the invention. One having ordinary skill
in the relevant art, however, will readily recognize that the invention
can be practiced without one or more of the specific details or with
other methods. The present invention is not limited by the illustrated
ordering of acts or events, as some acts may occur in different orders
and/or concurrently with other acts or events. Furthermore, not all
illustrated acts or events are required to implement a methodology in
accordance with the present invention.

[0017] All genes, gene names, and gene products disclosed herein are
intended to correspond to homologs from any species for which the
compositions and methods disclosed herein are applicable. Thus, the terms
include, but are not limited to genes and gene products from humans and
mice. It is understood that when a gene or gene product from a particular
species is disclosed, this disclosure is intended to be exemplary only,
and is not to be interpreted as a limitation unless the context in which
it appears clearly indicates. Thus, for example, for the genes disclosed
herein, which in some embodiments relate to mammalian nucleic acid and
amino acid sequences are intended to encompass homologous and/or
orthologous genes and gene products from other animals including, but not
limited to other mammals, fish, amphibians, reptiles, and birds. In
preferred embodiments, the genes or nucleic acid sequences are human.

DEFINITIONS

[0018] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of the
invention. As used herein, the singular forms "a", "an" and "the" are
intended to include the plural forms as well, unless the context clearly
indicates otherwise. Furthermore, to the extent that the terms
"including", "includes", "having", "has", "with", or variants thereof are
used in either the detailed description and/or the claims, such terms are
intended to be inclusive in a manner similar to the term "comprising."

[0019] The term "about" or "approximately" means within an acceptable
error range for the particular value as determined by one of ordinary
skill in the art, which will depend in part on how the value is measured
or determined, i.e., the limitations of the measurement system. For
example, "about" can mean within 1 or more than 1 standard deviation, per
the practice in the art. Alternatively, "about" can mean a range of up to
20%, preferably up to 10%, more preferably up to 5%, and more preferably
still up to 1% of a given value. Alternatively, particularly with respect
to biological systems or processes, the term can mean within an order of
magnitude, preferably within 5-fold, and more preferably within 2-fold,
of a value. Where particular values are described in the application and
claims, unless otherwise stated the term "about" meaning within an
acceptable error range for the particular value should be assumed.

[0020] As used herein, the term "safe and effective amount" or
"therapeutic amount" refers to the quantity of a component which is
sufficient to yield a desired therapeutic response without undue adverse
side effects (such as toxicity, irritation, or allergic response)
commensurate with a reasonable benefit/risk ratio when used in the manner
of this invention. By "therapeutically effective amount" is meant an
amount of a compound of the present invention effective to yield the
desired therapeutic response. The specific safe and effective amount or
therapeutically effective amount will vary with such factors as the
particular condition being treated, the physical condition of the
patient, the type of mammal or animal being treated, the duration of the
treatment, the nature of concurrent therapy (if any), and the specific
formulations employed and the structure of the compounds or its
derivatives.

[0021] As used herein "proteinuria" refers to any amount of protein
passing through a podocyte that has suffered podocyte damage or through a
podocyte mediated barrier that normally would not allow for any protein
passage. In an in vivo system the term "proteinuria" refers to the
presence of excessive amounts of serum protein in the urine. Proteinuria
is a characteristic symptom of either renal (kidney), urinary, pancreatic
distress, nephrotic syndromes (i.e., proteinuria larger than 3.5 grams
per day), eclampsia, toxic lesions of kidneys, and it is frequently a
symptom of diabetes mellitus. With severe proteinuria general
hypoproteinemia can develop and it results in diminished oncotic pressure
(ascites, edema, hydrothorax).

[0022] As used herein, the terms "podocyte disease(s)" and "podocyte
disorder(s)" are interchangeable and mean any disease, disorder,
syndrome, anomaly, pathology, or abnormal condition of the podocytes or
of the structure or function of their constituent parts.

[0023] The phrase "specifically binds to", "is specific for" or
"specifically immunoreactive with", when referring to an antibody refers
to a binding reaction which is determinative of the presence of the
protein in the presence of a heterogeneous population of proteins and
other biologics. For example, an antibody "specifically binds" or
"preferentially binds" to a target or epitope if it binds with greater
affinity, avidity, more readily, and/or with greater duration than it
binds to other targets. Thus, under designated immunoassay conditions,
the specified antibodies bind to a particular protein and do not bind in
a significant amount to other proteins present in the sample. Specific
binding to a protein under such conditions may require an antibody that
is selected for its specificity for a particular protein.

[0024] The terms "detecting", "detect", "identifying", "quantifying",
"measuring" includes assaying, quantitating, imaging or otherwise
establishing the presence or absence of the urinary proteins or other
disease indicators, and the like, or assaying for, imaging, ascertaining,
establishing, or otherwise determining the prognosis and/or diagnosis of
renal diseases, disorders or conditions.

[0025] "Patient" or "subject" refers to mammals and includes human and
veterinary subjects.

[0026] As used herein "a patient in need thereof` refers to any patient
that is affected with a disorder characterized by proteinuria. In one
aspect of the invention "a patient in need thereof refers to any patient
that may have, or is at risk of having a disorder characterized by
proteinuria.

[0027] As used herein, the terms "test substance" or "candidate
therapeutic agent" or "agent" are used interchangeably herein, and the
terms are meant to encompass any molecule, chemical entity, composition,
drug, therapeutic agent, chemotherapeutic agent, or biological agent
capable of preventing, ameliorating, or treating a disease or other
medical condition. The term includes small molecule compounds, antisense
reagents, siRNA reagents, antibodies, enzymes, peptides organic or
inorganic molecules, natural or synthetic compounds and the like. A test
substance or agent can be assayed in accordance with the methods of the
invention at any stage during clinical trials, during pre-trial testing,
or following FDA-approval.

[0028] As used herein the phrase "diagnostic" means identifying the
presence or nature of a pathologic condition. Diagnostic methods differ
in their sensitivity and specificity. The "sensitivity" of a diagnostic
assay is the percentage of diseased individuals who test positive
(percent of "true positives"). Diseased individuals not detected by the
assay are "false negatives." Subjects who are not diseased and who test
negative in the assay are termed "true negatives." The "specificity" of a
diagnostic assay is 1 minus the false positive rate, where the "false
positive" rate is defined as the proportion of those without the disease
who test positive. While a particular diagnostic method may not provide a
definitive diagnosis of a condition, it suffices if the method provides a
positive indication that aids in diagnosis.

[0029] As used herein the phrase "diagnosing" refers to classifying a
disease or a symptom, determining a severity of the disease, monitoring
disease progression, forecasting an outcome of a disease and/or prospects
of recovery. The term "detecting" may also optionally encompass any of
the above. Diagnosis of a disease according to the present invention can
be effected by determining a level of a polynucleotide or a polypeptide
of the present invention in a biological sample obtained from the
subject, wherein the level determined can be correlated with
predisposition to, or presence or absence of the disease. It should be
noted that a "biological sample obtained from the subject" may also
optionally comprise a sample that has not been physically removed from
the subject, as described in greater detail below.

[0030] As defined herein, "a therapeutically effective amount" of an agent
or compound (i.e., an effective dosage) means an amount sufficient to
produce a therapeutically (e.g., clinically) desirable result. The
compositions can be administered one from one or more times per day to
one or more times per week; including once every other day. The skilled
artisan will appreciate that certain factors can influence the dosage and
timing required to effectively treat a subject, including but not limited
to the severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases present.
Moreover, treatment of a subject with a therapeutically effective amount
of the compounds of the invention can include a single treatment or a
series of treatments. A "prophylactically effective amount" may refer to
the amount of an agent sufficient to prevent the recurrence or spread of
kidney diseases or disorders, particularly proteinuria, or the occurrence
of such in a patient, including but not limited to those predisposed to
kidney disease, for example those genetically predisposed to kidney
disease or previously exposed to environmental factors, such as for
example, alcohol or infectious organisms such as hepatitis virus. A
prophylactically effective amount may also refer to the amount of the
prophylactic agent that provides a prophylactic benefit in the prevention
of disease. Further, a prophylactically effective amount with respect to
an agent of the invention means that amount of agent alone, or in
combination with other agents, that provides a prophylactic benefit in
the prevention of disease.

[0031] The term "sample" is meant to be interpreted in its broadest sense.
A "sample" refers to a biological sample, such as, for example; one or
more cells, tissues, or fluids (including, without limitation, plasma,
serum, whole blood, cerebrospinal fluid, lymph, tears, urine, saliva,
milk, pus, and tissue exudates and secretions) isolated from an
individual or from cell culture constituents, as well as samples obtained
from, for example, a laboratory procedure. A biological sample may
comprise chromosomes isolated from cells (e.g., a spread of metaphase
chromosomes), organelles or membranes isolated from cells, whole cells or
tissues, nucleic acid such as genomic DNA in solution or bound to a solid
support such as for Southern analysis, RNA in solution or bound to a
solid support such as for Northern analysis, cDNA in solution or bound to
a solid support, oligonucleotides in solution or bound to a solid
support, polypeptides or peptides in solution or bound to a solid
support, a tissue, a tissue print and the like.

[0032] Numerous well known tissue or fluid collection methods can be
utilized to collect the biological sample from the subject in order to
determine the level of DNA, RNA and/or polypeptide of the variant of
interest in the subject. Examples include, but are not limited to, fine
needle biopsy, needle biopsy, core needle biopsy and surgical biopsy
(e.g., brain biopsy), and lavage. Regardless of the procedure employed,
once a biopsy/sample is obtained the level of the variant can be
determined and a diagnosis can thus be made.

[0034] The term "neutralizing" when referring to an targeted binding agent
such as an antibody relates to the ability of an antibody to eliminate,
or significantly reduce, the activity of a target antigen. Accordingly, a
"neutralizing" anti-uPAR antibody of the invention is capable of
eliminating or significantly reducing the activity of uPAR. A
neutralizing uPAR antibody may, for example, act by blocking the binding
of uPA to its receptor uPAR. By blocking this binding, the uPA mediated
plasminogen activation is significantly, or completely, eliminated.

[0035] "Active" or "activity" in regard to a uPAR polypeptide refers to a
portion of an uPAR polypeptide that has a biological or an immunological
activity of a native uPAR polypeptide. "Biological" when used herein
refers to a biological function that results from the activity of the
native uPAR polypeptide.

[0037] Antibodies of the present invention can be in any of a variety of
forms, including whole immunoglobulins, antibody fragments, single chain
antibodies which includes the variable domain complementarity determining
regions (CDR), and the like forms, all of which fall under the broad term
"antibody", as used herein.

[0038] The term "antigen binding fragment" refers to an antibody fragment
or portion of a full-length antibody, generally the variable region.
Examples of antigen binding fragments fragments of an antibody include
Fab, Fab', F(ab')2 and Fv fragments. Papain digestion of antibodies
produces two identical antigen binding fragments, called the Fab
fragment, each with a single antigen binding site, and a residual "Fc"
fragment, so-called for its ability to crystallize readily. Pepsin
treatment yields an F(ab')2 fragment that has two antigen binding
fragments that are capable of cross-linking antigen. Additional fragments
can include diabodies, linear antibodies, single-chain antibody
molecules, and multispecific antibodies formed from antibody fragments.

[0039] Single chain antibody ("SCA"), defined as a genetically engineered
molecule containing the variable region of the light chain, the variable
region of the heavy chain, linked by a suitable polypeptide linker as a
genetically fused single chain molecule. Such single chain antibodies are
also referred to as "single-chain Fv" or "sFv" antibody fragments.
Generally, the Fv polypeptide further comprises a polypeptide linker
between the VH and VL domains that enables the sFv to form the desired
structure for antigen binding. For a review of sFv see Pluckthun in The
Pharmacology of Monoclonal Antibodies 113: 269-315 Rosenburg and Moore
eds. Springer-Verlag, NY1 1994. Methods for producing sFvs are described,
for example, by Whitlow, et al., 1991, In: Methods: A Companion to
Methods in Enzymology, 2:97;

[0040] Another form of an antibody fragment is a peptide coding for a
single complementarity-determining region (CDR). CDR peptides ("minimal
recognition units") are often involved in antigen recognition and
binding. CDR peptides can be obtained by cloning or constructing genes
encoding the CDR of an antibody of interest. Such genes are prepared, for
example, by using the polymerase chain reaction to synthesize the
variable region from RNA of antibody-producing cells. See, for example,
Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2,
page 106 (1991).

[0041] The term "diabodies" refers to a small antibody fragments with two
antigen-binding sites, which fragments comprise a heavy chain variable
domain (VH) connected to a light chain variable domain (VL) in the same
polypeptide chain (VH-VL). By using a linker that is too short to allow
pairing between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, e.g., EP
404,097; or Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993).

[0043] Proteinuria can be primarily caused by alterations of structural
proteins involved in the cellular mechanism of filtration. The
pathophysiological causes of proteinuria can be divided in the following
major groups: (1) genetically determined disturbances of the structures
which form the "glomerular filtration unit" like the glomerular basement
membrane, the podocytes, or the slit diaphragm, (2) inflammatory
processes, either directly caused by autoimmune processes or indirectly
induced by microbes, (3) damage,of the glomeruli caused by agents, or (4)
as the final result of progressive tubulointerstitial injury finally
resulting in the loss of function of the entire nephron.

[0044] The central metabolism of a cell can determine its short- and
long-term structure and function. When a disease state arises, the
metabolism (i.e., the transportation of nutrients into the cells, the
overall substrate utilization and production, synthesis and accumulation
of intracellular metabolites, etc.) is altered in a way that may permit
the cell to adapt under the changing physiologic constraints. Diabetes
mellitus is a metabolic disease that also affects podocytes, key cells
that regulate glomerular filtration. A pathological role for a
cytoplasmic variant of cathepsin L enzyme as the biological instigator of
kidney filter dysfunction (proteinuria) and progression of renal disease
through cleavage of different types of critical podocyte target proteins.
Podocytes are highly differentiated cells that reside in the kidney
glomeruli. Their foot processes (FP) and interposed slit diaphragm (SD)
form the final barrier to protein loss. Podocyte injury is typically
associated with FP effacement and urinary protein loss.

[0045] In a healthy person, urinary protein excretion is less than 150
mg/day and consists mainly of filtered plasma proteins (60%) and tubular
Tamm-Horsfall proteins (40%). The main plasma protein in the urine is
albumin, constituting about 20% of daily protein excretion. In healthy
subjects, the daily amount of urinary albumin is less than 20 mg (13.8
mg/min). Proteinuria usually reflects an increase in glomerular
permeability for albumin and other plasma macromolecules. A 24-h urine
collection containing more than 150 mg of protein is considered
pathological. There are several basic types of proteinuria; for example,
glomerular, tubular, overflow, and exercise-induced. Glomerular
proteinuria is the most common form (around 90%). Low molecular weight
molecules, such as μ2-microglobulin, amino acids, and immunoglobulin
light chains, have a molecular weight of about 25 kDa (albumin is 69
kDa). These smaller proteins are readily filtered across the glomerular
filtration barrier and then fully reabsorbed by the proximal tubule. A
variety of diseases that affect tubular and interstitial cell integrity
impair the tubular reabsorption of these molecules. Some forms of
glomerular diseases are also accompanied by tubular injury and tubular
proteinuria.

[0046] Pathological processes, such as multiple myeloma with a production
of paraproteins, can result in increased excretion of low molecular
weight proteins into the urine, a process termed overflow proteinuria. In
this scenario, proteinuria results from the amount of filtered proteins
exceeding the reabsorptive capacity of the proximal tubule. Dynamic
exercise can also result in increased urinary excretion of proteins,
predominantly of plasma origin, during and following physical exercise. A
number of terms have been used to describe this phenomenon-post-exercise
proteinuria, athletic pseudonephritis, exercise proteinuria, or
exercise-induced proteinuria. Maximal rates of proteinuria occur
approximately 30 min after exercise, with a resolution toward resting
levels within 24-48 h. The magnitude of proteinuria varies from near
normal to heavy (47 g/day), with the greatest levels up to 100 times that
of rest observed after high-intensity exercise, such as a marathon. It is
noteworthy that post-exercise proteinuria is transient in nature and not
associated with any particular renal disease, raising the intriguing
possibility that at least some forms of proteinuria (e.g., post-exercise,
post-prandial, infection-associated) may reflect a normal, physiological
response of the human body.

[0047] Embodiments of the invention are directed to inhibiting both
soluble and membrane bound forms of urokinase receptor activation of
alpha V integrins. Both soluble as well as podocyte-membrane bound forms
of urokinase receptor can activate integrin alphaVbeta3
(αVβ3) as well as integrin alphaVbeta5
(αVβ5) in podocytes and cause renal disease. Urokinase
receptor (uPAR) signaling in podocytes has been recently shown to cause
glomerular disease. The soluble form of the urokinase receptor (suPAR)
can be deposited in the kidney and cause proteinuric renal disease. Like
endogenous podocyte uPAR, suPAR activates αvβ3
integrin in an outside in dependent fashion. uPAR is a
glycosylphosphatidylinositol (GPI)-anchored protein with three
extracellular domains. Cleavage of the GPI anchor generates suPAR. suPAR
has been found elevated in sera of patients with HIV, rheumatic or
neurological diseases, hematologic malignancies and epithelial tumors.
Proteinuria caused by uPAR-β3-integrin signaling can be prevented
and reduced by cyclo-RGDfV, a selective inhibitor of
αVβ3-integrin.

[0048] In a preferred embodiment, a composition comprises an agent which
specifically binds to αVβ3 and/or αVβ5 integrins
and modulates expression, function, signaling or combinations thereof.

[0051] In another preferred embodiment, an agent which specifically binds
to integrins αVβ3 and/or αVβ5 and modulates
signaling mediated events by these integrins is an antibody. VITAXIN®
(etaracizumab) is an example of an antibody that specifically binds to
integrin αVβ3. An example of an antibody which specifically
binds to integrins αVβ3 and αVβ5 is CNTO 95 (Trikha
M, et al., Int J. Cancer. 2004 Jun. 20; 110(3):326-35). CNTO 95 is a
fully human antibody that recognizes the alphaV family of integrins and
is to be less immunogenic in humans compared to chimeric or humanized
antibodies. CNTO 95 bound to purified αVβ3 and αVβ5
with a Kd of approximately 200 μM and to alphaV integrin-expressing
human cells with a Kd of 1-24 nM. In vitro, CNTO 95 inhibited human
melanoma cell adhesion, migration and invasion at doses ranging 7-20 nM.
(Trikha M, et al., Int J. Cancer. 2004 Jun. 20; 110(3):326-35). Other
preferred antibodies which specifically bind to integrins αVβ3
and/or αVβ5 include those having one or more (e.g., 1, 2, 3,
4, 5, or 6) of the complementarity determining regions of CNTO 95 or
etaracizumab.

[0052] In the studies, herein, the antibody was used to treat glomerular
kidney disease. Briefly, nephrosis was induced in rats by a single
injection of puromycin aminonucleoside (PAN) i.p. Two groups of rats were
formed. Group A (n=5) received only PAN, whereas the other group received
PAN plus escalating doses of CNTO 95 on days 1, 3, 5 and 7 before urine
was analyzed on day 8 (FIG. 1). Day 8 represents the peak time point for
proteinuria in this model. An approximate 27% reduction in proteinuria
was observed at this time. It is anticipated that CNTO 95 has about fifty
(50) fold more potency in humans over rat and thus a 27% reduction of
proteinuria in rats represents an excellent result.

[0053] In another preferred embodiment, a method of preventing or treating
kidney disease in vivo, comprises administering to a patient an agent in
a therapeutically effective amount, whereby the agent modulates the
expression, function or signaling of alphaV integrin molecules in vivo.
The agent is specific for binding to alpha V integrins and modulates the
expression, blocking the active binding site by molecules, such as, the
soluble and membrane bound forms of the urokinase receptor, the
activities or functions of alpha V integrin molecules, such as for
example, cell-to-cell interactions, inter- and intra-cellular signaling
and the like.

[0055] In another preferred embodiment, the agent specifically binds to
alphaVbeta3 (αVβ3) and alphaVbeta5 (αVβ5)
integrins.

[0056] In a preferred embodiment, the agent modulates or inhibits alphaV
integrin molecules expression, function and/or activity by about 5% as
compared to a normal control, preferably by about 10%, preferably by
about 50%, preferably by about 80%, 90%, 100%. Modulation of the, for
example, soluble urokinase receptor molecules expression or amounts
results in for example, a decrease in aV integrin activation and
treatment of renal diseases such as proteinuria.

[0057] In another preferred embodiment an agent inhibits or blocks
activated uPAR-β3-integrin signaling and uPAR-β5-integrin
signaling and podocyte FP hypermotility.

[0058] In another preferred embodiment, the composition comprises one or
more agents which modulate αV integrin expression, activity, and/or
function in vivo. For example, one agent directly inhibits αV
integrin activity. In another example, an agent directly inhibits binding
of uPAR to αV integrins or associated molecules which result in
changes to αV integrin signaling. In another preferred embodiment,
a mimetic of αVβ3 and αVβ5 ligand inhibits αV
activity or functions.

[0059] In another preferred embodiment, a combination of agents which
modulate αVβ3 and/or αVβ5 expression, function
and/or activity on are administered to a patient, for example, in the
treatment of a disease or disorder characterized by proteinuria and/or
podocyte diseases or disorders.

[0061] In another preferred embodiment, an agent which modulates
αVβ3 and αVβ5 integrin signaling is administered to
patients suffering from or pre-disposed to developing a podocyte disease
or disorder. Podocyte diseases or disorders include but are not limited
to loss of podocytes (podocytopenia), podocyte mutation, an increase in
foot process width, or a decrease in slit diaphragm length. In one
aspect, the podocyte-related disease or disorder can be effacement or a
diminution of podocyte density. In one aspect, the diminution of podocyte
density could be due to a decrease in a podocyte number, for example, due
to apoptosis, detachment, lack of proliferation, DNA damage or
hypertrophy.

[0062] In one embodiment, the podocyte-related disease or disorder can be
due to a podocyte injury. In one aspect, the podocyte injury can be due
to mechanical stress such as high blood pressure, hypertension, or
ischemia, lack of oxygen supply, a toxic substance, an endocrinologic
disorder, an infection, a contrast agent, a mechanical trauma, a
cytotoxic agent (cis-platinum, adriamycin, puromycin), calcineurin
inhibitors, an inflammation (e.g., due to an infection, a trauma, anoxia,
obstruction, or ischemia), radiation, an infection (e.g., bacterial,
fungal, or viral), a dysfunction of the immune system (e.g., an
autoimmune disease, a systemic disease, or IgA nephropathy), a genetic
disorder, a medication (e.g., anti-bacterial agent, anti-viral agent,
anti-fungal agent, immunosuppressive agent, anti-inflammatory agent,
analgesic or anticancer agent), an organ failure, an organ
transplantation, or uropathy. In one aspect, ischemia can be sickle-cell
anemia, thrombosis, transplantation, obstruction, shock or blood loss. In
one aspect, the genetic disorders may include congenital nephritic
syndrome of the Finnish type, the fetal membranous nephropathy or
mutations in podocyte-specific proteins, such as α-actin-4, podocin
and TRPC6.

[0063] In one aspect, the podocyte-related disease or disorder can be an
abnormal expression or function of slit diaphragm proteins such as
podocin, nephrin, CD2AP, cell membrane proteins such as TRPC6, and
proteins involved in organization of the cytoskeleton such as
synaptopodin, actin binding proteins, lamb-families and collagens. In
another aspect, the podocyte-related disease or disorder can be related
to a disturbance of the GBM, to a disturbance of the mesangial cell
function, and to deposition of antigen-antibody complexes and
anti-podocyte antibodies. In another aspect, the podocyte-related disease
or disorder can be tubular atrophy.

[0064] In a preferred embodiment, the podocyte-related disease or disorder
comprises proteinuria, such as microalbumiuria or macroalbumiuria. Thus,
in some preferred embodiments, one or more agents which modulate αV
integrin expression, function, activity, can be combined with one or more
other chemotherapeutic compounds which are used to treat any of the
podocyte diseases or disorders.

[0065] In another preferred embodiment, a method of preventing or treating
progressive glomerular disease comprises an agent which modulates
αV integrin expression, function, activity, and decreases
proteinuria levels to a clinically normal level. Proteinuria values or
levels can be measured by any typical assay, diagnostic or otherwise.

[0066] A wide variety of agents can be used to target αVβ3 and
αVβ5 integrins. These agents may be designed to target
signaling by having an in vivo activity which reduces the expression
and/or activity of αV and associated molecules. For example, the
agents may regulate αV molecules based on the cDNA or regulatory
regions, using for example, DNA-based agents, such as antisense
inhibitors and ribozymes, can be utilized to target both the introns and
exons of the αV genes as well as at the RNA level.

[0067] Alternatively, the agents may target αV molecules based on
the amino acid sequences including the propieces and/or three-dimensional
protein structures of αV molecules. Protein-based agents, such as
human antibody, non-human monoclonal antibody and humanized antibody, can
be used to specifically target different epitopes on αVβ3 and
αVβ5 molecules. Peptides or peptidomimetics can serve as high
affinity inhibitors to specifically bind to the active site of
αVβ3 and αVβ5, inhibiting the in vivo activity of
the αVβ3 and αVβ5, such as for example, signaling.
Small molecules may also be employed.

[0068] In addition to targeting αV molecules, agents may also be
used which competitively inhibit αV molecules by competing with the
natural ligands of αVβ3 and αVβ5.

[0069] Antibodies: In other embodiments of the invention described herein
relate to targeted binding agents that bind αV integrins and affect
αV function. Examples include, monoclonal antibodies that bind
αVβ3 and αVβ5 integrins and affect their function.

[0070] In another preferred embodiment, the invention relates to fully
human anti-αV antibodies which bind to both αVβ3 and
αVβ5 integrins with desirable properties from a therapeutic
perspective, including high binding affinity for αVβ3 and
αVβ5 integrins in vitro and in vivo.

[0071] In one embodiment, the invention includes antibodies that bind to
αVβ3 and αVβ5 integrins with very high affinities
(Kd). For example a human, rabbit, mouse, chimeric or humanized antibody
that is capable of binding αVβ3 and αVβ5 integrins
with a Kd less than, but not limited to, 10-5, 10-6, 10-7,
10-8, 10-9, 1040 or 10-11 M, or any range or value
therein Affinity and/or avidity measurements can be measured by
KINEXA.sup.TIv1 and/or BIACOR®.

[0072] One embodiment of the invention includes isolated antibodies, or
fragments of those antibodies, that bind to αVβ3 and
αVβ5 integrins. As known in the art, the antibodies can be,
for example, polyclonal, oligoclonal, monoclonal, chimeric, humanized,
and/or fully human antibodies. Embodiments of the invention described
herein also provide cells for producing these antibodies.

[0073] It will be appreciated that embodiments of the invention are not
limited to any particular form of an antibody or method of generation or
production. For example, the anti αVβ3 and αVβ5
antibody of the invention may be a full-length antibody (e.g., having an
intact human Fc region) or an antibody fragment (e.g., a Fab, Fab',
F(ab')2, Fv or Dab (Dabs are the smallest functional binding units
of human antibodies). In addition, the antibody may be manufactured from
a hybridoma that secretes the antibody, or from a recombinantly produced
cell that has been transformed or transfected with a gene or genes
encoding the antibody.

[0074] Other embodiments of the invention include isolated nucleic acid
molecules encoding any of the targeted binding agents, antibodies or
fragments thereof as described herein, vectors having isolated nucleic
acid molecules encoding anti-αVβ3 and αVβ5 integrin
antibodies or a host cell transformed with any of such nucleic acid
molecules. In addition, one embodiment of the invention is a method of
producing an anti-αVβ3 and αVβ5 antibody of the
invention by culturing host cells under conditions wherein a nucleic acid
molecule is expressed to produce the antibody followed by recovering the
antibody. It should be realized that embodiments of the invention also
include any nucleic acid molecule which encodes an antibody or fragment
of an antibody of the invention including nucleic acid sequences
optimized for increasing yields of antibodies or fragments thereof when
transfected into host cells for antibody production.

[0075] A further embodiment includes a method of producing high affinity
antibodies to αVβ3 and αVβ5 integrins by immunizing
a mammal with human αVβ3 and αVβ5 integrins, or a
fragment thereof, and one or more orthologous sequences or fragments
thereof.

[0076] In another embodiment, the invention includes an assay kit for
binding to αVβ3 and αVβ5 integrins in mammalian
tissues, cells, or body fluids to screen for kidney-related diseases. The
kit includes a targeted binding agent or an antibody of the invention
that binds to αVβ3 and αVβ5 integrins and a means
for indicating the reaction of the antibody with αVβ3 and
αVβ5 integrins, if present. In one embodiment, the antibody is
a monoclonal antibody. In another embodiment, the antibody that binds
αVβ3 and αVβ5 integrins is labeled. In still
another embodiment the antibody is an unlabeled primary antibody and the
kit further includes a means for detecting the primary antibody. In one
embodiment, the means for detecting includes a labeled second antibody
that is an anti-immunoglobulin. The antibody may be labeled with a marker
selected from the group consisting of a fluorochrome, an enzyme, a
radionuclide and a radiopaque material.

[0077] Other embodiments of the invention include pharmaceutical
compositions having an effective amount of a targeted binding agent or an
anti-αVβ3 and αVβ5 antibody of the invention in
admixture with a pharmaceutically acceptable carrier or diluent. In yet
other embodiments, the targeted binding agent or anti-αVβ3 and
αVβ5 antibody of the invention, or a fragment thereof, is
conjugated to a therapeutic agent. The therapeutic agent can be, for
example, a toxin or a radioisotope.

[0078] Yet another embodiment includes methods for treating diseases or
conditions associated with the uPAR mediated activation of
αVβ3 and αVβ5 integrins in a patient, by
administering to the patient an effective amount of a targeted binding
agent or an anti-αVβ3 and anti-αVβ5 antibody of the
invention. The targeted binding agent or anti-αVβ3 and
anti-αVβ5 antibody of the invention can be administered alone,
or can be administered in combination with additional antibodies or
chemotherapeutic drug or radiation therapy. For example, a monoclonal,
oligoclonal or polyclonal mixture of anti-αVβ3 and
anti-αVβ5 antibodies can be administered in combination with a
drug shown to inhibit a disease state or symptoms associated therewith.
The method can be performed in vivo and the patient is preferably a human
patient. In a preferred embodiment, the method concerns the treatment of
kidney disease comprises: podocyte diseases or disorders, proteinuria,
glomerular diseases, membranous glomerulonephritis, focal segmental
glomerulonephritis, minimal change disease, nephrotic syndromes,
pre-eclampsia, eclampsia, kidney lesions, collagen vascular diseases,
stress, strenuous exercise, benign orthostatic (postural) proteinuria,
focal segmental glomerulosclerosis (FSGS), IgA nephropathy, IgM
nephropathy, membranoproliferative glomerulonephritis, membranous
nephropathy, sarcoidosis, Alport's syndrome, diabetes mellitus, kidney
damage due to drugs, Fabry's disease, infections, aminoaciduria, Fanconi
syndrome, hypertensive nephrosclerosis, interstitial nephritis, Sickle
cell disease, hemoglobinuria, multiple myeloma, myoglobinuria, diabetic
nephropathy (DN), lupus nephritis, Wegener's Granulomatosis or Glycogen
Storage Disease Type 1.

[0079] In some embodiments, the targeted binding agent(s) or
anti-αVβ3 and anti-αVβ5 antibody(ies) of the
invention is administered to a patient, followed by administration of a
clearing agent to remove excess circulating antibody from the blood.

[0080] Nucleic Acid-based Agents: Nucleic acid-based agents such as
antisense molecules and ribozymes can be utilized to target both the
introns and exons of the αVβ3 and αVβ5 genes as
well as at the RNA level to inhibit gene expression thereof, thereby
inhibiting the activity of the uPAR mediated activation of these
molecules. Further, triple helix molecules may also be utilized in
inhibiting the αVβ3 and αVβ5 gene expression. Such
molecules may be designed to reduce or inhibit either the wild type
αVβ3 and αVβ5 gene, or if appropriate, the mutant
αVβ3 and αVβ5 gene. Techniques for the production
and use of such molecules are well known to those of skill in the art,
and are succinctly described below.

[0081] In another preferred embodiment, αVβ3 and
αVβ5 genes are modulated by targeting nucleic acid sequences
involved in the expression and/or activity of αVβ3 and
αVβ5 molecules. For example, regulatory regions would be a
target to decrease the expression of αVβ3 and αVβ5
or the regions which encode for the signaling domains.

[0082] Antisense RNA and DNA molecules act to directly block the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. Antisense approaches involve the design of
oligonucleotides that are complementary to a target gene mRNA. The
antisense oligonucleotides will bind to the complementary target gene
mRNA transcripts and prevent translation. Absolute complementarity,
although preferred, is not required.

[0083] A sequence "complementary" to a portion of an RNA, as referred to
herein, means a sequence having sufficient complementarity to be able to
hybridize with the RNA, forming a stable duplex; in the case of
double-stranded antisense nucleic acids, a single strand of the duplex
DNA may thus be tested, or triplex formation may be assayed. The ability
to hybridize will depend on both the degree of complementarity and the
length of the antisense nucleic acid. Generally, the longer the
hybridizing nucleic acid, the more base mismatches with an RNA it may
contain and still form a stable duplex (or triplex, as the case may be).
One skilled in the art can ascertain a tolerable degree of mismatch by
use of standard procedures to determine the melting point of the
hybridized complex.

[0084] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the AUG
initiation codon, should work most efficiently at inhibiting translation.
However, sequences complementary to the 3' untranslated sequences of
mRNAs have been shown to be effective at inhibiting translation of mRNAs
as well. Wagner (1994) Nature 372:333-335. For example, oligonucleotides
complementary to either the 5'- or 3'-untranslated, non-coding regions of
the human or mouse gene of urokinase receptor molecules could be used in
an antisense approach to inhibit translation of endogenous urokinase
receptor molecules mRNA.

[0085] In another preferred embodiment, the antisense approach can be used
to target negative regulators of αVβ3 and αVβ5
expression and/or function.

[0086] Oligonucleotides complementary to the 5' untranslated region of the
mRNA should include the complement of the AUG start codon. Antisense
oligonucleotides complementary to mRNA coding regions are less efficient
inhibitors of translation but could be used in accordance with the
invention. Whether designed to hybridize to the 5'-, 3'- or coding region
of target gene mRNA, antisense nucleic acids are preferably at least six
nucleotides in length, and are more preferably oligonucleotides ranging
from 6 to about 50 nucleotides in length. In specific aspects the
oligonucleotide is at least 10 nucleotides, preferably at least 17
nucleotides, more preferably at least 25 nucleotides and most preferably
at least 50 nucleotides.

[0087] Alternatively, antisense molecules may be designed to target the
translated region, i.e., the cDNA of the αVβ3 and
αVβ5 genes. For example, the antisense RNA molecules targeting
the full coding sequence or a portion of the mature murine urokinase
receptor molecules (Kirschke et al. (2000) Euro. J. Cancer 36:787-795)
may be utilized to inhibit expression of urokinase receptor molecules and
thus reduce the activity of its enzymatic activity. In addition, a full
length or partial urokinase receptor molecules cDNA can be subcloned into
a pcDNA-3 expression vector in reversed orientation and such a construct
can be transfected into cells to produce antisense polyRNA to block
endogenous transcripts of a uPAR, such as urokinase receptor molecules,
and thus inhibit uPAR's expression.

[0088] In vitro studies may be performed to quantitate the ability of the
antisense oligonucleotide to inhibit gene expression. It is preferred
that these studies utilize controls that distinguish between antisense
gene inhibition and nonspecific biological effects of oligonucleotides.
It is also preferred that these studies compare levels of the target RNA
or protein with that of an internal control RNA or protein. Additionally,
it is envisioned that results obtained using the antisense
oligonucleotide are compared with those obtained using a control
oligonucleotide. It is preferred that the control oligonucleotide is of
approximately the same length as the test oligonucleotide and that the
nucleotide sequence of the oligonucleotide differs from the antisense
sequence no more than is necessary to prevent specific hybridization to
the target sequence.

[0089] The oligonucleotides can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base moiety,
sugar moiety, or phosphate backbone, for example, to improve stability of
the molecule, hybridization, etc. The oligonucleotide may include other
appended groups such as peptides, or agents facilitating transport across
the cell membrane (See, e.g., Letsinger (1989) Proc. Natl. Acad. Sci.
U.S.A. 86:6553-6556) or the blood-brain barrier, hybridization-triggered
cleavage agents. See, e.g., Krol (1988) Bio Techniques 6:958-976 or
intercalating agents. See, e.g., Zon (1988) Pharm. Res. 5:539-549. The
oligonucleotide may be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent,
hybridization-triggered cleavage agent, etc.

[0091] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group consisting of, but not
being limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0092] In yet another embodiment, the antisense oligonucleotide comprises
at least one modified phosphate backbone selected from the group
consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or analog
thereof.

[0094] Ribozymes are enzymatic RNA molecules capable of catalyzing the
specific cleavage of RNA. The mechanism of ribozyme action involves
sequence specific hybridization of the ribozyme molecule to complementary
target RNA, followed by an endonucleolytic cleavage event. The
composition of ribozyme molecules should include one or more sequences
complementary to the target gene mRNA, and should include the well known
catalytic sequence responsible for mRNA cleavage.

[0095] While ribozymes that cleave mRNA at site specific recognition
sequences can be used to destroy target gene mRNAs, the use of hammerhead
ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations
dictated by flanking regions that form complementary base pairs with the
target mRNA. The sole requirement is that the target mRNA have the
following sequence of two bases: 5'-UG-3'. The construction and
production of hammerhead ribozymes is well known in the art.

[0098] Nucleic acid molecules to be used in triplex helix formation for
the inhibition of transcription should be single stranded and composed of
deoxynucleotides. The base composition of these oligonucleotides must be
designed to promote triple helix formation via Ho6gsteen base pairing
rules, which generally require sizeable stretches of either purines or
pyrimidines to be present on one strand of a duplex. Nucleotide sequences
may be pyrimidine-based, which will result in TAT and CGC triplets across
the three associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarity to a purine-rich
region of a single strand of the duplex in a parallel orientation to that
strand. In addition, nucleic acid molecules may be chosen that are
purine-rich, for example, contain a stretch of G residues. These
molecules will form a triple helix with a DNA duplex that is rich in GC
pairs, in which the majority of the purine residues are located on a
single strand of the targeted duplex, resulting in GGC triplets across
the three strands in the triplex.

Administration of Compositions to Patients

[0099] The compositions or agents identified by the methods described
herein may be administered to animals including human beings in any
suitable formulation. For example, the compositions for modulating
protein degradation may be formulated in pharmaceutically acceptable
carriers or diluents such as physiological saline or a buffered salt
solution. Suitable carriers and diluents can be selected on the basis of
mode and route of administration and standard pharmaceutical practice. A
description of exemplary pharmaceutically acceptable carriers and
diluents, as well as pharmaceutical formulations, can be found in
Remington's Pharmaceutical Sciences, a standard text in this field, and
in USP/NF. Other substances may be added to the compositions to stabilize
and/or preserve the compositions.

[0100] The compositions of the invention may be administered to animals by
any conventional technique. The compositions may be administered directly
to a target site by, for example, surgical delivery to an internal or
external target site, or by catheter to a site accessible by a blood
vessel. Other methods of delivery, e.g., liposomal delivery or diffusion
from a device impregnated with the composition, are known in the art. The
compositions may be administered in a single bolus, multiple injections,
or by continuous infusion (e.g., intravenously). For parenteral
administration, the compositions are preferably formulated in a
sterilized pyrogen-free form.

[0101] The compounds can be administered with one or more therapies. The
chemotherapeutic agents may be administered under a metronomic regimen.
As used herein, "metronomic" therapy refers to the administration of
continuous low-doses of a therapeutic agent.

[0102] Dosage, toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and it can
be expressed as the ratio LD50/ED50. Compounds that exhibit
high therapeutic indices are preferred. While compounds that exhibit
toxic side effects may be used, care should be taken to design a delivery
system that targets such compounds to the site of affected tissue in
order to minimize potential damage to uninfected cells and, thereby,
reduce side effects.

[0103] The data obtained from the cell culture assays and animal studies
can be used in formulating a range of dosage for use in humans. The
dosage of such compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity. The
dosage may vary within this range depending upon the dosage form employed
and the route of administration utilized. For any compound used in the
method of the invention, the therapeutically effective dose can be
estimated initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range that
includes the IC50 (i.e., the concentration of the test compound
which achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to more accurately determine
useful doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.

[0104] As defined herein, a therapeutically effective amount of a compound
(i.e., an effective dosage) means an amount sufficient to produce a
therapeutically (e.g., clinically) desirable result. The compositions can
be administered one from one or more times per day to one or more times
per week; including once every other day. The skilled artisan will
appreciate that certain factors can influence the dosage and timing
required to effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the general
health and/or age of the subject, and other diseases present. Moreover,
treatment of a subject with a therapeutically effective amount of the
compounds of the invention can include a single treatment or a series of
treatments.

Reduction of Proteinuria

[0105] In one aspect, the invention includes a method for reducing
proteinuria or urinary albumin in a subject. In this method, the subject
is administered a sufficient amount of an agent that targets and
modulates the function of αVβ3 and/or αVβ5
integrins such that proteinuria or concentrations of urinary albumin are
reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or more percent
post-treatment. The agent can be a monoclonal antibody that specifically
binds αVβ3 and/or αVβ5 integrins (e.g., CNTO 95).
Alternatively, the agent can inhibit uPARand/or suPAR binding to the
urokinase receptor or can specifically bind to these molecules thus
preventing their binding or deposition in the podocytes.

[0106] Treatment of Subjects with Abnormal suPAR levels: The invention
includes a method including a step of administering an agent that targets
and modulates the function of αVβ3 and/or αVβ5
integrins to a subject having abnormally high serum suPAR (e.g., greater
than 3500, 3600, 3700, 3800, 390, 4000, 4500, or 5000 pg/ml serum as
determined by ELISA or other assays). In one embodiment, the agent is a
monoclonal antibody that specifically binds αVβ3 and/or
αVβ5 integrins (e.g., CNTO 95). This method can also include a
step of determining whether the subject has an abnormal serum suPAR level
(with or without renal disease or symptoms) and/or a step of selecting
and/or modulating the dosing of the agent that targets and modulates the
function of αVβ3 and/or αVβ5 integrins according to
the subject's suPAR levels (e.g., lower dose for patients with lower but
still high suPAR levels, and titrating the dose according to a subject's
response).

[0107] The invention also includes a method for reducing pathologic levels
of activated αVβ3 and/or αVβ5 integrins on human
podocytes by contacting the cells with an agent that targets and
modulates the function of αVβ3 and/or αVβ5
integrins such as CNTO 95.

[0108] In another aspect, the invention features a method including the
step of administering an agent that targets and modulates the function of
αVβ3 and/or αVβ5 integrins such as CNTO 95 or a
like antibody to a subject with proteinuria but not cancer and/or a
subject that is also being treated with other drugs for kidney disease
(e.g., ACE inhibitors, angiotensin receptor blockers, diuretics,
steroids, calcium carbonate, calcitriol, sevelamer, erythropoietin,
darbepoetin, iron, and/or vitamin D) or for drugs that can address any
possible side effects of CNTO 95 (e.g., acetaminophen, ibuprofen, or
other pain or fever reducers; antihistamines; and/or anti-nausea
medications). The step of administering an agent that targets and
modulates the function of αVβ3 and/or αVβ5
integrins may be performed by the methods described herein as well as by
more specifically directing the kidney using a renal infusion system such
as the BENEPHIT® system (AngioDynamics).

[0109] Reduction of Activated αVβ3 on Podocytes: Also within
the invention is a method of reducing the function of activated
αVβ3 on podocytes, e.g., in a subject with abnormally high
levels of activated a Vf33 on his/her podocytes (e.g., more than 100,
200, 300, 400, or 500% of normal levels). This method can include the
step of contacting the podocytes with an agent that targets and modulates
the function of αVβ3 and/or αVβ5 integrins such as
CNTO 95.

[0110] While various embodiments of the present invention have been
described above, it should be understood that they have been presented by
way of example only, and not limitation. Numerous changes to the
disclosed embodiments can be made in accordance with the disclosure
herein without departing from the spirit or scope of the invention. Thus,
the breadth and scope of the present invention should not be limited by
any of the above described embodiments.

[0111] All documents mentioned herein are incorporated herein by
reference. All publications and patent documents cited in this
application are incorporated by reference for all purposes to the same
extent as if each individual publication or patent document were so
individually denoted. By their citation of various references in this
document, Applicants do not admit any particular reference is "prior art"
to their invention.